Gene,91 (1990) 135-138 Elsevier

135

GENE 03605

Expression of the gene encodingfirefly luciferase in insect cells using a baculovirusvector (Recombinant DNA; Spodopteraf.ig@erda; Photinus;Autogmphu; reporter enzyme; regulatory signals; targeting)

Seyed

E. Hasnaln and Blta Nakbal

NationalInstituteof Immunology.New Delhi 110067 (India) Received by J.-P. Lecocq: 2 May 1989 Revised: 4 September1989 Accepted: 9 November 1989 and 6 April 1990

SUMMARY

A cDNA encoding the fnefly luciferase [Photinusluciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing), EC 1.13.12.71was cloned downstream from the polyhedrin gene promoter of Autogmphucal#imtk~ nuclear polyhedrosis virus and expressed in Spodopterafigiperdu clone-9 cells. Synthesis of luciferase (Luc) was accurately measured in insect cells growing in a 96,well plate, by a simple, rapid, nonradioactive, inexpensive and sensitive method based on fogging of x-ray film. Luc was produced in a coordinate fashion during virus infection. The Luc synthesized in insect cells was not secreted into the medium but was contained within the cell. Our findings suggest that Luc can be used as a superior reporter enzyme for molecular genetic analyses of baculovirus regulatory signals involved in high level expression of foreign genes, protein processing, targeting and stability in insect cells.

INTRODUmION

The baculovirus expression vector system utilising AcNPV and Sf9 cells is a very widely used heterologous expression system (Cameron et al., 1989; Luckow and Summers, 1988; Miller et al., 1986).The level of expression ranges from l-800 pg/ml and the synthesized protein is identical to its authentic counterpart in almost all respects

Correspondenceto: Dr. S.E. Hasnain, Gene Expression Laboratory, National Instituteof Immunology,Shahid Jeet Singh Marg,New Delhi 110067 (India) Tel. 686-3004,ext. 301; Fax 91116862125.

Abbreviations:aa, aminoacid(s); AcNPV,Autogruphacul@nicu nuclear polyhedrosisvirus;bp, base pair(s);CAT, chloramphenicolacetyl transferase; cDNA, DNA complementary to mRNA; kb, kilobase or 1000bp; Luc, luciferase enzyme; /UC,Luc-encodinggene; moi, multiplicity of infection; nt, nucleotide(s); pus, polyadenylationsite; pi, post infection; SB, Spodopterujiwgiperdu clone-9insect cells; tsp,transcription start point(s); VA&C,recombinantbaculoviruswhich expresses the luc gene; wt, wild type. 0378-l119/90/$03.500 1990ElsevierSciencePublishersB.V. (BiomedicalDivision)

(Luckow and Summers, 1988). Although insect cells are capable of most post-translational modifications such as glycosylation, phosphorylation, myristylation, accurate protein folding, secretion, targeting to organelles, etc., the regulatory signals involved in these processes are yet to be identified. Gene fusions, where a reporter gene not present in the host system is fused to regulatory sequences, are excellent tools for molecular genetic analyses. Luc is one such reporter enzyme that catalyses the light-producing chemical reaction of bioluminescent organisms. The extensively characterised insect Luc, isolated from the firefly Photinus pyralis, has been widely used as a reporter in eukaryotic cells. The aim of the present study was to clone a luc gene in a baculovirus vector and express it in Sf9 cells for possible use as reporter enzyme. These cells have zero background activity for Luc and those infected with VA&C can be assayed for Luc by a simple, rapid, sensitive, inexpensive and non-isotopic method.

136 vAc lu_.c.c3

EXPERIMENTAL AND DISCUSSION

(a) Expression of luc in insect cells Physical map ofpAcluc, the recombinant transfer vector, which was used to construct the recombinant vAcluc virus is shown in Fig. 1. Northern-blot analysis of total RNA isolated from infected Sf9 cells at 54 h pi, revealed two well-defined mRNA species corresponding to approx. 1.8 kb and 2.'/kb in vAcluc-infected cells (Fig. 2), but not in AcNPV or mock-infected cells. The presence of two transcripts suggests initiation and/or termination of transcription from tspP-pasP (polyhedrin) and tspL-pasL (luc gene) (Fig. 1). The polyhedrin tsp is about 40 nt upstream from the BamHl cloning site in pAc373 (Summers and Smith, 1987). The iuc fragment also has its own initiation and termination signals (deWet et al., 1987) which can easily explain the presence of an approx. 1.8-kb transcript. On the other hand, if the polyhedrin terminator is utilised the mRNA will be approx. 2.7 kb in length. The levels ofthe two luc transcripts are identical which indicates that initiation of transcription from the polyhedrin promoter proceeds without any bias for homologous or heterologous tsp and perhaps shows no bias for recognition of the termination signals either. It is noteworthy that translation of one or both of the iuc mRNAs will still result in a single Luc polypeptide.

(b) Assay for Lue Luc enzyme requires luciferin, ATP and oxygen as substrates. The absolute requirement for ATP is characteristic for firefly Luc (doWer et al., 1985). Our Luc assay was

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designed keeping in mind the fact that enough ATP is available within the intracellular pool, and luciferin can easily permeate the cytoplasmic membrane (deWet et al., 1987). The enzymatically active Luc produced in Stx) cells (Fig. 3) apparently requires much less luciferin as opposed to 217/AM in mammalian cells, or 400/AM in plant cells (Ow et al., 1986). AcNPV-infected cells did not exhibit any detectable endogenous Luc activity, thus indicating total absence of background Luc in Sf9 cells. The commonly used assay (deWet et al., 1985), though very sensitive, meet-

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Fig. I. Partial restriction map ofplasmid pAcluc. A 1.8-kbBamHI fragment ofpDO432 (Ow et al., 1986)containing the intron-less P.pyralb luc gene (eDNA) was cloned into pAc373 (Summers and Smith, 198,/)to construct the recombinant transfer vector pAc&c. Sf9 cells were maintained and used for transfoction,plaqueassayand virus propagationas describedby Summersand Smith (198,/). Co-transfection of Sf9 cells with the recombinanttransfer vector pAcluc and wt AcNPV DNA Generatedthe recombinant vAc/uc virus, which had a replacement of the polybedrin protein-coding region by the [uc eDNA. Virus vArJucrepresents isolate No, 6 (i.e. vAc/uc6), which is one ofthe three different isolates characterised, and this was used for infection at all times, unless otherwise indicated. The integration of luc at the polyhedrin locus in a correct orientation relative to the polyhedrin Sane promoter is shown. The BamHl site in pAcY/3 was designated as + I, Distance in nt is given in parentheses. The tsp for polyhedrin and luc (tspP, tspL) and the pas for polyhedrin and luc (pasP. pasL) are indicated. Fig, 2. Northern-blot analysis of total RNA in infected cells. RNA was isolated from cells infected with AcNPV, and vAcluc and from mock-infected cells, as described by Summers and Smith (1987), denatured with formamide, fractionated on 1.4% agnrose gel, transferred to nylon membrane and probed with a nick-translated (Maniatis et al., 1982) 1.8-kb BamHl fragment ofpDO432 (Ow et al., 1986) containing the luc gene. H/mdlll digest of 3 DNA was used as standard; sizes are indicated (in kb) on the left margin. The luc.specific transcripts are clearly absent in AcNPV or mock-infected cells.

137 sures the peak intensity of the resulting flash of light while simultaneously generating a background noise produced by the lurninometer, thereby decreasing the signal-to-noise ratio. In earlier reports of Luc synthesised in m~mmalian cells it was shown that Luc was about a few orders ofmagnitude more sensitive than commonly used reporter enzymes, such as p-galactosidase (Rodriguez et al., 1988), or CAT (deWet et al., 1987). Moreover, its synthesis could be monitored, using a luminometer, from as low as 5 × 106 cells. We could initially (at 0.5 moi) detect the presence of Luc in 200 cells (Fig. 3), however, the enzyme activity could be easily monitored from a single cell using a higher titre of the virus (at 10-20 moi). Comparison of the sfg-synthesised Luc with commercially available (Sigma Chemical Co., USA) Luc standards (not shown) indicated that at about 60 h pi, Luc levels reached approx. 150 pg/106 cells. It is, therefore, apparent that insect cells are making fairly large amounts ofthis enzyme. A more detailed analysis of Luc synthesised in Sf9 cells at different times after infection is shown in Fig. 4, which is typical for genes under the polyhedrin gene 18h

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promoter. The fogging of x-ray film by the flash of light, produced as a result of Luc activity, is a very simple assay for Luc and this makes it an attractive reporter enzyme for insect cells. (¢) Celinlu Iocalimfion of Luc An attempt was made to localise the Luc produced in infected cells by assaying for Luc in vAc/uc:u~ected cell supematant and pellet (Fig. S). Mock-in£ected cells (Fig. 5A) or infected cell supematant (Fig. 5D) did not register any fogging of x-ray film. Lue synthesised in SI~ cells is, therefore, not secreted into the culture medium but retained within the cell (Fig. 5C). Furthermore, vAc/ucinfected cells did not exhibit any cell-surface fluorescence, but gave intense intracellular fluorescence reactions localised within the peroxisome-like organdie in the lantern organ (deWet et al., 1987), where the high oxygen tension ensures efficient availability of substrates for this enzyme. Keller et al. (1987) demonstrated the peroxisomal targeting of a recombinant Luc synthesised in mammalian cells and subsequently showed that the peroxisomal sorting domain was present within the last 12 aa at the C-terminal end (Gould et al., 1987). Recently, Miyazawa et al. (1989) have identified the targeting signal for another peroxisomal enzyme, rat-liver acyl-coenzyme A oxidase. Interestingly, a highly conserved Ser-Lys-Leu motif which is part of the cell-sorting domain, is also present at the C-terminal end of the firefly Luc and few other peroxisomal enzymes.

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138 as is evident from the expression of a Luc fusion protein in Escherichia coli (deWet et al., 1985). The simple, rapid, sensitive and inexpensive assay described in this paper makes Luc a superior reporter enzyme for understanding the molecular signals involved in processing, targeting and stability ofheterologous proteins in insect cells. These cells are increasingly being used as host for synthesis of secretory glycoproteins. Although the general conclusion from previous studies that these cells cannot process the immature N-linked oligosaccharide to an endo-/~-N-acetyl-D-glucosaminidase H-resistant form has been refuted (Jarvis and Summers, 1989), much is yet to be known about the secre tory pathway of these cells. Though Luc synthesised in Sf9 cells does not enter the secretory pathway, it is retained intracellularly. This should provide an excellent model system for understanding the precise nature of the proteinsorting domain and the accompanying molecular machinery involved in the movement of proteins ~between organelles (Kelly, 1987). This will also aid in the construction of new transfer vectors with improved levels of expression of foreign gene.

ACKNOWLEDGEMENTS

We wish to express our sincere thanks to Prof. G.P. Talwar, Director, National Institute of Immunology, for his encouragement and support of this research; Dr. Max D. Summers, Texas A&M University, U.S.A., for Sf9 cells, virus and viral vectors; Dr. Stephen H. Howell, University of California at San Diego, CA, for pDO432, and Dr. M. Bustos, Texas A& M University, for introducing us to the baoulovirus system. This investigation was supported by a grant (BT/TF/03/026/007/88) to SEH from the Department of Biotechnology, Government of India.

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